A novel gene from a secondary metabolism gene cluster is required for microsclerotia formation and virulence in Verticillium dahliae
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Verticillum dahliae is a notorious plant pathogenic fungus, which can cause wilt disease on hundreds of plant species. The genome of V. dahliae has been sequenced and annotated, but incorrect gene annotations and unidentified transcribed regions persist. Using RNA-Seq combined with reverse-transcription PCR, we identified a novel transcribed gene, Nag1, located in a cluster of putative secondary metabolic genes whose roles remain unknown. Functional analysis of Nag1 by dsRNA-mediated gene silencing revealed that loss of Nag1 significantly decreased fungal growth and conidial production. In addition, Nag1-silenced mutants exhibited obvious defects in microsclerotia formation and fungal virulence. Consistent with the reduction in microsclerotia formation, melanin production and expression of genes involved in melanin biosynthesis were markedly reduced in Nag1-silenced mutants. Overall, our data suggest that Nag1 acts as an important regulator of fungal development, microsclerotia formation and secondary metabolism in V. dahliae.
KeywordsNovel transcript region RNAi-mediated silencing Melanin biosynthesis
Open reading frame
Potato dextrose agar
Verticillium dahliae Kleb., the causal agent of Verticillium wilt disease, is a notorious pathogenic fungus that can infect more than 200 plant species and result in enormous economic loss all over the world (Fradin and Thomma 2006; Klosterman et al. 2009). Due to the pathogen’s ability to colonize the xylem and the longevity of microsclerotia, no effective control methods for Verticillium wilt disease are available (Wilhelm 1955). Microsclerotia are melanized resting structures formed by V. dahliae that play critical roles during the disease cycle (Puhalla and Bell 1981; Klimes et al. 2015). Upon encountering a suitable host, microsclerotia germinate into hyphae and infect plants through the roots (Klimes et al. 2015). However, the mechanisms underlying microsclerotia development in V. dahliae remain to be elucidated.
Recent advances in sequencing technology have greatly facilitated functional genomics research. RNA-Seq is a powerful technology for examining changes in transcriptional profiles under various conditions (Wang et al. 2009), and can also be used to improve annotation of gene models and detect novel transcribed regions (Roberts et al. 2011). In addition, alternative splicing and RNA editing events can also be identified by taking advantage of RNA-Seq technology (Pan et al. 2008; Peng et al. 2012).
RNA interference (RNAi), also known as gene silencing, is a natural phenomenon observed in plants, fungi, and animals. This phenomenon plays diverse roles in genomic defense, heterochromatin formation, and gene regulation (Agrawal et al. 2003; Matzke and Birchler 2005). RNAi, a post-transcriptional mode of regulation, decreases the expression of target genes by degrading mRNA with homologous double-stranded RNA (dsRNA) (Fire et al. 1998). This method has been developed as a useful tool for genetic manipulation, and has been successfully used for analysis of gene functions in all kinds of organisms. In contrast to gene knockout technology, RNAi can be used to characterize the functions of multi-copy and essential genes. In filamentous fungi, three well-known RNAi strategies can be used to manipulate the expression of a target gene: expression of a hairpin RNA, production of dsRNA by convergent transcription, and direct introduction of dsRNA into fungal cells (Khatri and Rajam 2007; Nakayashiki and Nguyen 2008; Li et al. 2010). In general, the former two methods (expression of a hairpin RNA and production of dsRNA by convergent transcription) are much more efficient. However, expression of hairpin RNA is time-consuming and not well suited for high-throughput analysis, whereas production of dsRNA by convergent transcription can be easily manipulated and facilitates large-scale functional analysis. The functions of 37 calcium-signaling genes in Magnaporthe oryzae were characterized using a convergent transcription approach (Nguyen et al. 2008), and ten endo-β-1,4 xylanase genes were simultaneously silenced in M. oryzae using a similar strategy (Nguyen et al. 2011). Furthermore, RNAi method (expression of a hairpin RNA) has been used to analyze the functions of the chorismate synthase encoded gene (Singh et al. 2010) and the C2H2 transcription factor gene Vta2 (Tran et al. 2014) in the near-diploid fungus Verticillium longisporum, indicating that RNAi is suitable for use in Verticillium species.
Secondary metabolites are often bioactive but not essential for fungal survival (Keller et al. 2005). The most famous secondary metabolite is penicillin, which has saved hundreds of millions of human lives (Bennett and Chung 2001). By contrast, some fungal secondary metabolites are poisonous, such as Fumonisin B1 in Fusarium verticillioides (Wang et al. 1991; Nelson et al. 1993). Polyketides, non-ribosomal peptides, terpenes, and indole alkaloids are representative examples of fungal secondary metabolites, and the genes responsible for their biosynthesis are usually arranged in clusters (Keller et al. 2005; Fox and Howlett 2008). Melanin, a type of polyketide, is one of the most common secondary metabolites produced by fungi, and it has functions in cell wall stability and facilitating survival in hostile environments (Nosanchuk and Casadevall 2003). In fungi, melanin is synthesized via two main pathways: the L-3-4-dihydroxyphenylalanine pathway, in which dihydroxyphenylalanine is oxidized by laccase and polymerized into melanin, as in Cryptococcus neoformans (Eisenman et al. 2007; Eisenman et al. 2009); and the 1, 8-dihydroxynaphthalene (DHN) pathway, which is operative in V. dahliae (Bell et al. 1976; Stipanovic and Bell 1976; Wheeler et al. 1976). Melanin is generated during microsclerotia formation in V. dahliae, and several genes involved in this process have been identified (Duressa et al. 2013; Xiong et al. 2014; Fang et al. 2019). Other pathogenic fungal species, including M. oryzae (Soanes et al. 2012) and Sporothrix schenckii (Romero-Martinez et al. 2000), also produce melanin through the DHN pathway.
In this study, we identified a previously overlooked gene (Nag1) in the V. dahliae genome and characterized its functions by the dsRNA-induced gene silencing approach. The resultant Nag1-silenced mutants exhibited obvious defects in fungal growth, conidial production, fungal virulence, and microsclerotia formation. Additionally, the production of melanin was significantly reduced in Nag1-silenced mutants, and the expression levels of genes involved in this process were also reduced.
Identification of a new annotated gene from RNA-Seq data
Based on these findings, we considered this novel transcribed region as a new annotated gene, Nag1, consisting of two exons and one intron (exon 1: 441660–441,756, intron: 441604–441,659, exon 2: 440708–441,603). Nag1 encodes a protein of 330 amino acids with no detectable structural motifs other than a DUF3425 (protein of unknown function) domain (Additional file 1: Figure S1 and Fig. 1d). Remarkably, the complete sequence of Nag1 is also present in the genome of V. dahliae JR2, with 100% sequence identity.
Identification of Nag1 homologs in other fungal species
RNAi-mediated down-regulation of Nag1 impairs fungal growth
Nag1 is involved in cell wall integrity
To determine the role of Nag1 in cell wall integrity, all strains were inoculated onto standard CM plates (control) or CM containing NaCl, KCl, MgSO4, Calcofluor White (CFW), and Congo Red. At 10 days post inoculation (dpi), colony diameters were significantly reduced on the supplemented plates relative to the control plates, especially on NaCl and KCl supplemented plates, which reduced growth diameter of Nag1-silenced mutants and wild type by over 50% compared with the strains grown on the CM plates, respectively (Additional file 4: Figure S4). Nag1-S3 and Nag1-S6 exhibited slightly higher resistance to NaCl, KCl, and MgSO4 than the wild-type strain, which showed higher relative growth in Nag1-silenced mutants (over 40% relative growth in NaCl, KCl and 92% relative growth in MgSO4 relative to the CM plates) compare to wild type (36.6% relative growth in NaCl, KCl and 87% relative growth in MgSO4 relative to the CM plates) (Additional file 4: Figure S4a, b). These results indicated that silencing of Nag1 slightly decreased sensitivity to osmotic stresses. We also investigated the sensitivity of all three strains to oxidative stress (5% or 15% H2O2), but observed no distinguishable difference in the size of the inhibition zone (Additional file 4: Figure S4c), indicating that Nag1 is not required for resistance to H2O2.
Silencing of Nag1 dramatically decreases microsclerotia formation
We also observed the early development of microsclerotia by scanning electron microscopy. In the wild-type strain, massive mycelia formed and covered the cellulose membrane, and aerial mycelia formed at 36 hpi. By 48 hpi, we could observe formation of swollen mycelia, which gathered to generate microsclerotia between 60 and 72 hpi (Fig. 5b). By contrast, Nag1-S3 did not form the aerial mycelia and swollen mycelia from 36 to 72 hpi. It took 60 h for Nag1-S3 to cover the cellulose membrane, and aerial conidiophores developed at 72 hpi (Fig. 5b). Thus, the developmental processes of microsclerotia were delayed about 2 days in Nag1-S3 relative to the wild-type strain, largely resulting from defects in fungal growth. These results suggested that Nag1 plays an important role in microsclerotia formation (Fig. 5).
Silencing of Nag1 decreases fungal virulence
Silencing of Nag1 significantly decreases melanin biosynthesis
Silencing of Nag1 decreases expression of secondary metabolism genes
As a result of technical advances and cost reductions in sequencing, more and more species have been sequenced. To improve genome annotations and reveal specific features of various isolated strains, an increasing number of species have also been re-sequenced, yielding a large body of sequence data and annotated gene models. However, achieving correct gene annotation remains a challenge. Generally, prediction of gene models is performed by machine annotation with FGENESH, GENEID, and other bioinformatic tools (Cuomo et al. 2007; Klosterman et al. 2011). However, many of these annotations lack transcriptional evidence, and some gene model predictions are inaccurate, especially in the case of species-specific and non-conserved genes. Additionally, it is relatively difficult to distinguish novel genes and untranslated regions by bioinformatic prediction alone. However, annotation of gene model predictions could potentially be improved using multiple methods performed in F. graminearum (Wong et al. 2011; Zhao et al. 2013). Our previous work also revised an erroneous annotation of gene model in V. dahliae based on sequence alignment and reverse-transcription PCR (Yang et al. 2013).
RNA-Seq is a useful method for revealing the transcriptional changes in eukaryotes (Cloonan et al. 2008; Metzker 2009; Davidson et al. 2012), and it is also an efficient tool for improving the gene models, discovering non-coding RNAs, and identifying novel transcript regions (Graveley et al. 2010; Li et al. 2011; Ren et al. 2012; Zhao et al. 2013). In this study, using bioinformatic methods and reverse-transcription PCR, we discovered a novel transcribed region in RNA-Seq data that was predicted to encode a novel annotated gene (Nag1) of V. dahliae. Interestingly, candidate Nag1 orthologs were only found in A. niger, and the functions of these proteins remain to be identified. Functional analysis of Nag1 by dsRNA-mediated gene silencing revealed that Nag1 is involved in fungal growth, conidial production, melanin biosynthesis, microsclerotia formation, and fungal virulence.
Nag1 is located in a secondary metabolism gene cluster in V. dahliae, and highly conserved homolog sequences of the whole gene cluster were found in the genome of V. alfalfae (Xiong et al. 2016). However, synteny of this whole gene cluster could not be found in A. niger and F. verticillioides. Although the homologs of VDAG_07930 and VDAG_07931 are adjacent in the A. niger genome, the Nag1 homolog is not located between them, and the VDAG_07928 homolog is located in another region (Additional file 6: Figure S6). In addition, the homologs of VDAG_07930, Nag1, and VDAG_07931 in the A. niger CBS genome do not belong to a secondary metabolism gene cluster predicted by the J. Craig Venter Institute (http://www.jcvi.org/smurf/index.php). Synteny of this secondary metabolism gene cluster region was only observed between V. dahliae and V. alfalfae, although single-gene homologs could be found in other fungal species. Regarding this specific secondary metabolism gene cluster, the backbone gene VDAG_07928 was the only PKS/NRPS hybrid gene predicted in the V. dahliae genome, whereas different numbers of PKS/NRPS hybrid genes were observed in other fungal species (Khaldi et al. 2010). Importantly, functional analysis of ACE1, the homolog of VDAG_07928 in M. oryzae, revealed that it is required for recognition of the fungus by resistant hosts (Böhnert et al. 2004). And the results show that the product synthesized by the PKS/NRPS hybrid gene cluster of ACE1 is involved in plant-pathogen recognition. Interestingly, deletion of ACE1 in M. oryzae does not dramatically affect fungal pathogenicity, indicating that the secondary metabolite synthesized by ACE1 and its gene cluster is not a key pathogenicity factor. In addition, a PKS/NRPS hybrid gene in Trichoderma virens is also involved in induced systemic resistance, and the putative secondary metabolite generated by the gene cluster is important for the fungus–plant interaction (Mukherjee et al. 2012). In this study, silencing of Nag1 led to a significant reduction of gene expression in the same gene cluster, and the backbone gene VDAG_07928 was almost not expressed in Nag1-silenced mutants. However, we observed a reduction in the pathogenicity of Nag1-silenced mutants, indicating that secondary metabolites synthesized by VDAG_07928 and its cluster might be important for fungal pathogenicity. These results suggested that PKS/NRPS hybrid genes in different fungal species may play different roles.
Phenotypic observations revealed that Nag1-silenced mutants exhibited a significant reduction in microsclerotia formation, but Nag1 was not differentially expressed during microsclerotia formation as shown in Fig. 1a. As mentioned above, silencing of Nag1 resulted in dramatic reduction of gene expression in the same secondary metabolism cluster, and our previous transcriptomic analysis revealed that genes in this cluster from VDAG_07920 to VDAG_07929 were significantly up-regulated during microsclerotia formation, although VDAG_07930 and VDAG_07931 were excluded (Xiong et al. 2014). Thus, the reduction of microsclerotia formation in Nag1-silenced mutants might result from Nag1-induced down-regulation of gene expression from VDAG_07920 to VDAG_07931. In addition, the Nag1-silenced mutants shared many phenotypes with the ΔVdMcm1 mutant, e.g., the reduction in fungal growth, microsclerotia formation, fungal virulence, and melanin accumulation. Importantly, our previous work showed that the whole gene cluster from VDAG_07920 to VDAG_07931, including Nag1, was regulated by VdMcm1. These results suggested that many defects of the ΔVdMcm1 mutant may be derived from down-regulation of this secondary metabolism gene cluster. Interestingly, the Nag1-silenced mutants also showed many common phenotypes with the VdPR3 (VDAG_09942 encodes a predicted protein) deletion mutants in V. dahliae which also showed reduction in fungal growth, conidial production, microsclerotia formation, melanin accumulation, and fungal pathogenicity (Zhang et al. 2015). Additionally, it had been reported that an APSES transcription factor, vst1, led to strong downregulation of the gene cluster comprising VDAG_07920 - VDAG_07931, and deletion of this gene showed significant defects in microsclerotia formation and melanin production (Sarmiento-Villamil et al. 2018).
In this study, we identified and characterized the functions of a novel annotated gene, Nag1, in V. dahliae. Our results showed that Nag1 is involved in fungal growth, microsclerotia formation, cell wall integrity, melanin biosynthesis and fungal virulence which will facilitate the genetic studies of pathogenicity and microsclerotia formation in V. dahliae.
Strains and media
V. dahliae XS11, isolated from smoke tree (Cotinus coggygria), was used as the wild-type strain throughout this study (Wang et al. 2013). The strain V. dahliae Ls17 (ATCC accession MYA-4575), isolated from lettuce in California (CA), was kindly provided by Dr. Steven J. Klosterman (USDA-ARS). Generally, all strains were grown on potato dextrose agar (PDA) plates at room temperature. To test the growth rate and conidia production, the strains were grown on PDA plates and the experiment was repeated three times. To assay the conidial germination, conidia were harvested from cultures grown in fresh liquid complete medium (CM) (Dobinson et al. 1997) by filtration through two layers of Miracloth (Calbiochem, Germany) and then grown for 2 days in liquid basal medium (BM) (Neumann and Dobinson 2003) with 150 rpm. The percentage of conidial germination was measured by randomly recording 100 conidia, and the experiment was repeated three times. For DNA or RNA extractions, cultures were prepared in liquid CM. For stress response assays, all strains were grown on CM plates with or without 1.2 M NaCl, 1.2 M KCl, 0.5 M MgSO4, 10 μg/mL CFW and 100 μg/mL Congo Red. To assay the sensitivity to H2O2, a 5 mm filter paper disk was placed on the CM plates containing 106 spores and then 5 μL 5% or 15% (v/v) H2O2 were dropped onto the filter paper disk as described previously (Molina and Kahmann 2007), and the experiment was repeated three times with three repetitions. The growth inhibition induced by the H2O2 was measured after 2 days of incubation at room temperature. To stimulate microsclerotia formation in vitro, 105 spores/mL conidial suspension was spread over cellulose membranes (pore size = 0.22 μm) which were overlaid onto the BM as described previously (Xiong et al. 2014). To observe the microsclerotia formation, a part of the membrane was observed with scanning electron microscopy. To reveal the accumulation of melanin, mycelia were grown in Czapek-Dox salts medium (3 g/L sodium nitrate, 0.5 g/L KCl, 0.5 g/L MgSO4·7H2O, 1 g/L K2HPO4, 0.01 g/L FeSO4·7H2O) (Bailey et al. 1997) supplemented with 3% sucrose.
Construction of silencing vectors and transformation
The putative ORF of Nag1 was amplified with primers 5/6. A 417 bp fragment of Nag1 was amplified from the cDNA of V. dahliae XS11 with primers 7/8, which was then ligated into the pSilent-Dual1 (pSD1, which was bought from The Fungal Genetics Stock Center) that was digested with SmaI, named pSD1-Nag1. The pSD1-Nag1 was introduced into the protoplasts of V. dahliae XS11 (Wang et al. 2013). Transformants were selected on TB3 medium (1 L: 3 g yeast extract, 3 g casamino acids, 20% sucrose, 0.7% agar for solid medium) with 50 μg/mL geneticin (Sangon, China).
Sequence and data analysis
The RNA-Seq coverage data were visualized using Integrative Genomics Viewer (Robinson et al. 2011). Sequence of Nag1 was acquired from V. dahliae genome database in Joint Genome Institute (JGI) (Klosterman et al. 2011). The putative ORFs were predicted by ORF Finder in National Center for Biotechnology Information (NCBI). The Nag1 homologs in other fungal species were identified using the BLASTP and BLASTN programs with default parameters in each genome database in JGI. Amino acid sequence alignments were performed with ClustalX2.0 (Larkin et al. 2007). Phylogenetic tree was constructed with MEGA 6.0 (Tamura et al. 2013) using neighbor-joining method and the bootstrap test was replicated 1000 times. The domain architecture was visualized using InterPro (http://www.ebi.ac.uk/interpro/). Alignments of amino acid and nucleotide sequences were visualized using BioEdit (Hall 1999). Difference of measurement data was compared with single factor analysis of variance at P ≤ 0.05 levels by using SPSS.
For pathogenicity tests, conidia were harvested from 7-day-old cultures grown in liquid CM by filtration through two layers of Miracloth and resuspended at 106 conidia/mL in sterile distilled water. One-year-old smoke tree seedlings were inoculated by dipping their roots in the conidial suspensions for 10 min. Control plants were mock-inoculated with sterile distilled water. All inoculated smoke tree seedlings were then replanted into the soil. Five smoke tree seedlings were tested per strain and the experiment was repeated three times. The inoculated plants were photoed after 45 days.
Total RNA was extracted using Trizol Reagent (Invitrogen, USA) and further purified with a PureLink RNA Mini Kit (Ambion, USA) and PureLink Dnase (Invitrogen, USA) according to the manufacturer’s instructions. For qRT-PCR assays, cDNA was synthesized using oligo (DT)18 primer and SuperScriApt III Reverse Transcriptase (Invitrogen, USA). qRT-PCR reactions were performed using SuperReal PreMix Plus (Tiangen, China) with the Applied Biosystems 7500 Real-Time PCR system. The β-tubulin gene was used as an internal reference for all qRT-PCR analyses. Relative expression levels were calculated using 2-△△Ct method (Livak and Schmittgen 2001). All primers used in this study were listed in Additional file 7: Table S1.
Ethical approval and consent to participate
DX performed the experiments, analyzed the data and wrote the manuscript. YW and CT designed the experiments and revised the manuscript. All authors read and approved the final manuscript.
This study was funded by the Fundamental Research Funds for the Central Universities (BLX201703).
Consent for publication
The authors declare that they have no competing interests.
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